The present invention relates to a vessel for withdrawing blood, and the blood withdrawn should especially be used for stabilizing and analyzing nucleic acids.
When blood is taken, it is normally collected in vessels which already contain anticoagulants such as heparin, citrate or EDTA. The blood is thereby prevented from coagulating. The blood samples obtained thereby can be stored at suitable temperatures for a long time. This way of obtaining blood has, however, considerable drawbacks when nucleic acids such as (m)RNA or DNA are to be analyzed. For such purposes the nucleic acids contained in the sample should optimally be stabilized already at the moment of withdrawal, i.e. a degradation of the existing nucleic acids should be prevented, but also the new synthesis of mRNA.
This objective of a stable storage of the nucleic acids contained in the sample material, i.e. from the moment of withdrawal, has not been achieved yet in practice for the following reasons:
Cells contain nucleases, enzymes, which destroy nucleic acids as soon as they come into contact with the substrates thereof (RNA, DNA). The effect of cellular and extracellular nucleases is normally under physiological control as long as the cells are in their normal environment. The withdrawal of blood effects more or less strong changes in the nucleic acids contained in the cells. Nucleases are then released within the cells and/or by the lysis of cells to the outside. Moreover, nucleic acids are synthetized more or less strongly. In particular the long-term storage of blood leads to aging and destruction of the cells.
Another problem arising in the long-term storage of blood samples obtained according to standard withdrawal methods is the considerable change in the sample material. Such changes, e.g. strong lysis of cells, may have the effect that the standard methods for isolating nucleic acids no longer function in an adequately efficient way.
Apart from the problems regarding a stable storage of nucleic acids contained in the sample material, further difficulties arise in the conventional method for withdrawing blood. The conventional anticoagulants are often not separated efficiently enough during isolation of nucleic acids and interfere with in the subsequent analysis of nucleic acids, e.g. in the case of amplification by means of PCR (polymerase chain reaction). Heparin is e.g. a generally known inhibitor of PCR.
Finally, the question arises in the quantitative analysis of nucleic acids how the whole method ranging from sampling to the measurement of nucleic acids can be controlled under standardized conditions. Ideally, a quantitatively and qualitatively defined standard nucleic acid should already be added to the sample material during withdrawal and should be subjected to the whole process of sampling and determination. This can also not be accomplished with the conventional withdrawal systems.
A further drawback of conventional blood withdrawal is the risk of transferring infectious material because manual process steps have so far been needed for the isolation of nucleic acids. Contact with potentially infectious germs cannot be ruled out.
In the literature there is described a method in which the blood sample is mixed with guanidinium salt directly after withdrawal from a patient (EP 0 818 542 A1). In this method the guanidinium salt is present in powder form to thereby exploit the increased stability of the guanidinium salt. This method, however, has serious drawbacks because the salt, for instance, must first dissolve in the added blood. This dissolution process depends, in particular, on the temperature and cannot be controlled because of the nontransparent sample material used. The use of a corresponding product for diagnostic medical purposes is thus very problematic.
Furthermore, nucleases are extremely active enzymes which can only be inhibited under extremely denaturing conditions. Denaturation depends on the concentration of the guanidinium salt in solution. An inhibiting concentration of guanidinium salt in solution does not exist in the cited method right from the beginning. Thus, there is an uncontrolled degradation of nucleic acids during the dissolution process. Moreover, in this method the addition of reducing agents is omitted, without which an efficient inhibition, in particular of RNases, is not ensured (see Example no. 5).
Moreover, the sample prepared in this way cannot directly be used for the further nucleic acid isolation on glass surfaces. Moreover, the use of guanidinium salt powder does not permit the addition of internal nucleic acid standards. Such standards are mandatory for process control and exact quantification.
The present invention has been based on the technical problem of providing a vessel for withdrawing blood which does not have the drawbacks of the prior art. In particular, it should be possible to subject the sample taken with the vessel directly to the standard methods for analyzing nucleic acids without the need for further sample preparation steps.
According to the invention this problem is solved by a vessel for withdrawing blood, the vessel containing an aqueous solution comprising the following components:
a guanidinium salt;
a buffer substance;
a reducing agent; and/or
a detergent.
The vessel of the invention has the following advantages: 1. Blood is already lysed at the moment of withdrawal in that the withdrawal vessel already contains a nucleic acid-stabilizing substance in solution. 2. The nucleic acid-stabilizing substance is composed such that the sample material, in particular the nucleic acids contained therein, are directly stabilized upon contact with the solution. 3. In contrast to all of the former standard withdrawal systems, such as EDTA or heparin-containing withdrawal vessels, the stabilized sample need no longer be handled as infectious material. 4. The nucleic acid-stabilizing substance is composed such that the sample material can directly be used in subsequent isolating methods. 5. The nucleic acid-stabilizing substance can be separated during subsequent isolation so efficiently that an inhibition of PCR is not observed. 6. The nucleic acid-stabilizing substance may have added thereto an internal standard. This permits the control of the whole method from the moment of sampling up to the detection of nucleic acids.
The withdrawal vessel mentioned under item 1 is a conventional blood withdrawing vessel (small tube) which has introduced thereinto a defined volume of a nucleic acid-stabilizing substance. The small tube is then preferably subjected to a defined vacuum which guarantees that only a specific volume of blood can flow thereinto during withdrawal. The small tube can be handled by conventional blood-taking methods. The solution contained in the tube contains the following reagents in a specially preferred embodiment: Guanidinium thiocyanate, Triton-X-100, dithiothreitol and a suitable buffer system, such as citrate, Tris or HEPES. In the described composition the solution is compatible with the vacuum tube. This solution can be stored in the vacuum tube without any problems and without any impairment of the desired stabilizing function. The whole system presents no problems, in particular to blood donors, and is safe during sampling.
The solution containing the guanidinium salt, the buffer substance, the reducing agent and/or the detergent is stable in storage and converts the supplied and freshly taken blood into a material which is also stable in storage and can directly be subjected to the standard nucleic-acid analysis kits (e.g. those of Roche or Qiagen).
Guanidinium thiocyanate and/or guanidinium chloride are preferred as guanidinium salt.
Preferably, the guanidinium salt is present in a concentration of 2.0 to 8.0 M. Tris or citrate is preferred as the buffer substance, the exact pH being preferably adjusted with HCl. Further possible buffers are however HEPES, MOPS, citrate and phosphate buffer, such as PBS.
The buffer concentration is preferably between 10 and 300 mM, particularly preferably between 10 and 100 mM.
Triton-X-100 is preferred as the detergent. Further possible detergents are NP40, Tween 20, polydocanol or other detergents.
The detergent concentration is preferably at 5 to 30% (w/v), particularly preferably at 10 to 20% (w/v).
DTT is preferred as the reducing agent, but xcex2-mercaptoethanol, TCEP (Tris(2-carboxyethyl)phosphine) or other reducing agents can also be used.
The preferred concentration of the reducing agent is at 0.1 to 10% (w/v), particularly preferred are 0.5 to 2% (w/v).
The pH of the solution is preferably at 3.0 to 9.0, particularly preferably at 4.0 to 7.5, particularly preferably at 5 to 6.
The pH of the solution is in particular chosen such that a pH ranging from 5.0 to 7.6 is set after addition of the sample material. Particularly preferred is a pH between 6.3 and 6.9 (see Example no. 8).
A particularly preferred solution preferably contains 4 M guanidinium thiocyanate, 45 mM Tris/HCl, 18%, preferably 15% (w/v) Triton-X-100, 0.8% (w/v) DTT and has a pH of 6.0.
In a further preferred embodiment the volume for receiving the blood sample has a negative pressure which can be adjusted such that a previously determined blood volume is sucked into the vessel after a blood vessel has been pierced. Correspondingly evacuated vessels are available on the market.
The vessel which contains the blood taken can then immediately be subjected to further analyses or, however, may be stored for a long period of time (up to several days) without any disadvantages for the quality of the sample.
In the method of the invention the freshly taken blood is directly contacted in the blood withdrawing vessel with the above-described solution so that all processes which might change the nucleic acid pattern of the sample are immediately stopped. Therefore, the data determined at a later time with respect to the detected nucleic acids very accurately represent the actual state at the time of blood withdrawal, i.e. both with respect to the quantities and the types of nucleic acids.
Preferably, the blood amount taken is 0.1 to 4 times the solution fed into the vessel. The solution is preferably 0.5 to 5.0 ml. Thus the final concentration of guanidinium salt after blood addition is at 1.0 to 5 M, preferably at 1 to 3 M, particularly preferred are 2-3 M (see Example 7).
The vessel according to the invention is preferably used for blood withdrawal when the blood sample is to be used for analyzing nucleic acids.
The use of the above-mentioned solution as a component of the described withdrawal system solely guarantees the immediate lysis of the cells and the simultaneous stabilization of the sample by immediate inactivation of the nucleases. Surprisingly, the blood sample obtained thereby can be stored even at room temperature or higher for several days. Moreover, the withdrawal system guarantees a contamination-free and non-infectious handling ranging from sampling via nucleic acid isolation to analysis. In the conventional methods of nucleic acid isolation, additional handling steps have so far been required (e.g. the transfer of the blood sample taken into the reagents for nucleic acid isolation, etc.), which entails an additional risk of infection.
The sample obtained with the blood withdrawing system is compatible with all of the conventional standard methods of nucleic acid isolation. Particular attention should here be paid to methods which are based on the binding of nucleic acids to glass surfaces, but also sequence-specific binding to complementary nucleic acid and solvent-based extraction methods.
Thus the invention as described consists of a blood withdrawing system which is conceived such that the following conditions are satisfied. 1. Controlled sampling and simultaneous stabilization of the nucleic acids (DNA, RNA) contained in the sample material. 2. Sampling in which the use of anticoagulants can be completely omitted. 3. The sample obtained by way of the above-described blood withdrawing system can be used in a universal manner in all of the known systems for isolating nucleic acids. 4. The blood withdrawing system is stable in storage.
Additionally, it has surprisingly been found that the sample obtained by way of the described withdrawal system can be stored in the vessel for a long period of time without degradation of the nucleic acids (see Examples 2, 3, 7, 8).